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Anti-tumor T cell as well as preparation method and anti-tumor drug thereof

An anti-tumor drug, tumor cell technology, applied in anti-tumor drugs, animal cells, drug combinations, etc., can solve the problem of no malignant brain tumor clinical data and other problems

Inactive Publication Date: 2015-05-20
深圳市中美康士生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the small size of resected tumor samples and the limited availability of TILs, there are no clinical data on the use of TILs in the treatment of malignant brain tumors

Method used

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  • Anti-tumor T cell as well as preparation method and anti-tumor drug thereof
  • Anti-tumor T cell as well as preparation method and anti-tumor drug thereof
  • Anti-tumor T cell as well as preparation method and anti-tumor drug thereof

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preparation example Construction

[0056] The second aspect of the present invention is to provide a method for preparing anti-tumor T cells as described above, comprising the following steps:

[0057] Step 1. Preparation of total tumor RNA: synthesize tumor total RNA from cultured tumor cells in vitro;

[0058] Step 2. Preparation of mature tumor total RNA-DC: the tumor total RNA obtained in step 1 and the isolated dendritic cell DC were introduced into the DC by electrotransduction, and then cultured overnight in the culture medium to obtain mature tumor total RNA-DC. RNA-DC;

[0059] Step 3. Co-culture the mature tumor total RNA-DC obtained in step 2 with T cells to obtain the anti-tumor T cells according to claim 1.

[0060] In the above preparation method, preferably, the tumor cell line in step 1 is from human or mouse tumor cells, more preferably from but not limited to the highly invasive mouse astrocytoma cell line KR158 and melanoma cell line B16F10OVA.

[0061] In the above preparation method, prefe...

Embodiment

[0072] 1. Materials and methods:

[0073] 1. Cell culture and ttRNA preparation from mouse tumor cell lines:

[0074] Mouse cytokines IL-4, IL-7, IL-12, IL-15, IL-21, GM-CSF and CD40L were purchased from PEPROTECH (Los Angeles, New Jersey), and IL-2 (Aldesleukin) was purchased from PROLEUKIN ( San Diego, California). Mouse T cells were cultured in RPMI containing 5% FBS supplemented with 100 U / ml penicillin / streptomycin, 1 mM L-glutamine, 55 uM β-ME, 1 mM sodium pyruvate, 0.1 mM NEAA, 10 mM HEPES (Invitrogen Corporation). In this example, the highly invasive mouse astrocytoma model and melanoma IC tumor model are preferred, that is, from NF1 - / - :p53 - / - The astrocytoma cell line KR158 in C57BL / 6-mice, and the melanoma cell line B16F10OVA from mice cultured in DMEM with 10% FBS. All cells were cultured at 37°C in a 5% CO2 humidified incubator. Lipopolysaccharide (LPS) was purchased from Sigma (St. Louis, MO). The total tumor RNA (total tumor RNA, ttRNA) from KR158 and B...

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Abstract

The invention provides a method for preparing an anti-tumor T cell in vitro through DC (dendritic cell, namely antigen presenting cell), wherein the anti-tumor T cell is a tumor total RNA-loaded DC amplification and enrichment anti-tumor T cell. The invention also provides a preparation method of the anti-tumor T cell and an anti-tumor drug or inoculation vaccine taking the anti-tumor T cell as an active ingredient. According to the invention, a specific anti-tumor T cell is amplified in vitro by taking the tumor total RNA-loaded DC as a platform, a small amount of tumor total RNA is amplified to an enough amount in vitro so as to realize clinical application, and the in-vivo anti-tumor effect in an IC (intracranial) tumor-bearing model is evaluated, including how the mature state of DC and the cell factor combination adjust the differentiation of the anti-tumor T cell as well as the synergistic anti-tumor effect thereof in a preclinical experiment model.

Description

technical field [0001] The invention relates to an anti-tumor T cell and a preparation method thereof, as well as an anti-tumor drug or vaccination which uses the anti-tumor T cell as an active ingredient. Background technique [0002] The direct use of in vitro expanded anti-tumor T cells is based on two facts: 1) patients with malignant brain tumors have strong systemic immunosuppression, which seriously hinders the effect of anti-tumor vaccination methods; Compared with adoptive cell transfer (ACT) using in vitro expanded anti-tumor T cells including tumor infiltrated lymphocytes (TIL) or genetically engineered peripheral blood lymphocytes (PBL) Can more effectively eradicate residual tumor cells in melanoma patients (Dudley ME, Yang JC, Sherry R, ​​Hughes MS, Royal R, et al. (2008)Adoptive cell therapy for patients with metastatic melanoma: evaluation of intensive myeloablative chemoradiation Preparative regimens. J Clin Oncol 26:5233-5239; Morgan RA, Dudley ME, Wunderl...

Claims

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Application Information

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IPC IPC(8): C12N5/0783A61K35/13A61K39/00A61P35/00
Inventor 杨世成
Owner 深圳市中美康士生物科技有限公司
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